Project Summary The primary goal of this competitive grant renewal remains to identify genes and regulatory non-coding sequences that generate species-specific differences in development and organization of the cerebral cortex, particularly association areas, such as the prefrontal cortex. The prefrontal cortex is considered the crucible of human cognitive capacities with well-documented neuroanatomical connectivity and unique cellular features that are thought to be undermined in neuropsychiatric disorders and hijacked by drugs of abuse. We start with the assumption that basic principles of cortical development in all mammals are remarkably similar. It is, however, reasonable to expect important quantitative (e.g. the number of neurons, tempo and sequence of cellular events) and qualitative changes (e.g. the introduction of new neuronal subtypes, elaboration of synaptic connections and addition of functionally specialized cortical areas) since primates split from the rodent lineage about 100 million years ago. Thus, our strategy has been to study in parallel developmental events in the rodent (mouse), non-human primate (macaque) and human embryonic telencephalon by using the most advanced molecular and cell biological methods available, including comparative high-resolution mRNA-sequencing, in utero and ex utero gene manipulation (Loss or Gain of function) and heterologous transplantation of neural stem cells. We will complete and further augment our ongoing high-resolution mRNA-sequencing, confirm results by qRT-PCR, and gain further insight through bioinformatics network analysis in three species, which was initiated in the first cycle of this grant (Aim #1). Then, based on our discovery of several primate-specific cortical neuronal subtypes, we now plan to identify their genetic determinants by performing lineage analysis and use of ex-utero electroporation to overexpress and/or knockdown selected genes to examine their interactions and identify downstream transcription factors (Aim #2). Finally, we will proceed to the next stage of this research by identifying regulatory changes driving human and nonhuman primate-specific gene expression and binding sites on downstream targets followed by comparison of ChIP-seq data to differential gene expression (Aim #3). Although the proposed research is extremely time-consuming, logistically difficult and costly, it is realistic based on our published record and our progress, which shows that we succeeded to establish the unique facilities, master and modify essential methodology and already have obtained a substantial amount of feasibility data. We argue that abnormal genesis and initial formation of the evolutionarily novel, human- specific traits of the cerebral cortex may be particularly vulnerable to genetic mutations and environmental influences each of which alone, or in combination, can give rise to elusive neuropsychiatric disorders and neuronal response to prenatal exposure to drugs of therapy and abuse.